Ascorbate-isoquercetin compositions

ABSTRACT

The present invention relates to novel compositions containing ascorbic acid with an increased bioavailability of this vitamin. These compositions are useful as food supplements possessing preventive properties against damages of human organs, including skin, tissues and cells due to oxidative stress or damages.

The present invention relates to novel compositions containing ascorbicacid with an increased level of its active form. These compositions areuseful as food supplements possessing preventive properties againstdamages of human tissues, including skin cells due to oxidative stress.

In vivo ascorbic acid (vitamin C) exists in three forms:

a) as an ascorbate in form of an ascorbate monoanion,

b) as the reversibly oxidised form of a free radical, calledsemidehydroascorbic acid which could be reversibly oxidised todehydroascorbic acid or reversibly reduced to ascorbate monoanion, and

c) as dehydroascorbic acid (oxidised form of semidehydroascorbic acid).

Only ascorbate possesses specific vitamin C activity: as a cofactor forenzymes. Observed physiological activities of semidehydroascorbic acidand dehydroascorbic acid formed in vivo from ascorbate are considered tobe based on their reversible reductions to ascorbates, (Buettner,1993—Dharival et al., 1991; Welch et al., 1995 Washko et al., 1993). Thesecond form of ascorbic acid, semidehydroascorbic acid (ascorbate freeradical) participates in univalent redox systems, (Bors et al. 1995),that is in the antioxidant defence activity. This means,semidehydroascorbic acid participates most likely in free radicalscavenging activities. According to Gordon (1996, p. 270), “ascorbateappears to be the most important non-protein antioxidant in plasma”.Ascorbic acid is absorbed from the gastrointestinal tract in the form ofascorbic acid. Dehydroascorbic acid is reduced to ascorbic acid forgastrointestinal absorptions (Rose et al., 1988).

Structures of body tissues are susceptible to damages caused by theoxidative stress, e.g. by the accumulation of reactive oxygen speciesduring ageing, chronic environmental stress, inflammations or generalmetabolic dysfunctions. The role of free radicals and reactive oxygenspecies in aetiology of human diseases (e.g. cancer, atherosclerosis,rheumatoid arthritis, inflammatory bowel diseases, immune systemdysfunctions, brain function decline, connective tissue dysfunctions) iswell established (for a recent review see: Gordon, 1996). Uncontrolledgeneration of free radicals, especially chronic exposure to reactiveoxygen species leads to chronic intracellular damages, to oxidativestress and premature ageing. Cells of the human body possess metabolicantioxidant defences which are supported by dietary antioxidants. Theearly observations of the antioxidant defence metabolic processesinvolved vitamin C and flavonoids (Bezssonoff, 1926; Bentsath et al.,1936; Bensath et al., 1937; Blanc and Von der Muehl, 1967). Ascorbicacid is not only important non-protein antioxidant in human plasma(Gordon, 1.c.) but it increases (Skaper et. al., 1997) thecytoprotective activities of quercetin and rutin. Skaper and co-authors(1997) have shown, for instance, that quercetin protects connectivetissue and specifically skin cells (e.g. fibroblasts, keratinocytes, andendothelial cells) from this type of damages. Other authors havedemonstrated protective effect of flavonols on cardiovascular andnervous system, their role as chemoprotective agents in carcinogenesis.

Oxidation of the ascorbate in the human body by xenobiotics often leadsto the accumulation of semidehydroascorbic acid or dehydroascorbic acidin organs where these forms interfere with the regular metabolism. Asascorbate is a cofactor for eight isolated enzymes (carrying outcollagen synthesis, carnitine synthesis, peptide amidation, tyrosinemetabolism, and catecholamine synthesis) the decrease of theconcentration of ascorbate in body tissues and fluids may leads toserious metabolic dysfunctions.

The possibilities to protect ascorbic acid in vivo were based on veryearly observations of Szent-Györgyi group mentioned above that theascorbic acid activity in humans and guinea pigs is intensified by thegreat group of “vegetable dyes, the flavons or flavonols”. It has beenknown that flavonoids are contributing to the maintenance of theconcentration of the administered ascorbate in adrenals, kidneys,spleen, and the liver of the organisms investigated and improve theantiscorbutic effect of the dosages of ascorbate used (Papageorge andMitchell, 1948; Cotereau et al., 1948; Crampton and Lloyd, 1950; Douglasand Kamp, 1959; Blanc and Von der Muehl, 1967; Zloch, 1973).

The mechanism of this effect, called “the vitamin C-economisingfunction” of some flavonoids (“facteur d'economie de L'acide ascorbique”of Bezssonoff, 1926 and 1927) has been recognised in many laboratories.For example, Harper et al., 1969, found that, among flavonoids tested,flavonols the have strongest ability to inhibit ascorbic acid oxidationin near neutral solutions (pH 5–7). Harper et al. (1.c.) also pointedout that the presence of free hydroxyl groups at carbon atoms-3, 7, 3′,and 4′ in a flavonol molecule improves the antioxidative effect of theflavonol molecule, this means, it inhibits ascorbate oxidation moreeffectively.

But there was neither an effective method nor a useful orally applicableformulation leading to an increased level of active ascorbate in humantissue.

Accordingly, there was a need for a composition useful for theprotection of the orally administered ascorbic acid and enhancement ofvitamin activity in the tissues.

Now it has been found that isoquercetin effectively inhibits ascorbateoxidation. The maintenance of the reduced form of ascorbic acid byisoquercetin maintains ascorbic acid level in body tissues and fluids.

This effect perhaps may be explained in that isoquercetin not only showsthree free hydroxyl groups mentioned by Harper (1.c.), more exactly,hydroxyl groups attached to carbon atoms 7, 3′, and 4′, but also aglucopyranoside moiety with additional four free hydroxyl groups0-attached to the carbon 3 of isoquercetin. Therefore, the increasedeffectivity of ascorbate protectioin may be caused by the fact thatisoquercetin contains a glucose molecule. This glucose molecule seems tobe the reason why isoquercetin is able to use the sodium-dependentglucose transport pathway of the intestinal brush-border membrane in itsabsorption process (Gee et al., 1998). Experiments have also shown thatthe absorption of isoquercetin is better than that of pure aglycone.

Earlier pharmacokinetic studies with isoquercetin anticipated resultsobtained and explained by Gee at al,. 1.c., by having shown excellentabsorption rate and bioavailability of isoquercetin (Hollman and Katan,1997).

It has been found that ascorbate is not only able to regenerate oxidisedflavonols by reducing them (Yamasaki et al., 1997) but also to protectquercetin (aglycone of the isoquercetin) against oxidative degradationand to maintain the antiviral properties of quercetins (Vrijsen et al.,1988).

This means, there is a synergistic effect between isoquercetin andascorbate in human tissue leading to higher effectivities of both,ascorbate and isoquercetin.

For isoquercetin these activities are as follows:

it has shown anthypertensive properties, (Kameda et al., 1987); itinhibits the biosynthesis and release of prostaglandin-like substances(Chanh et al. 1986); it produces dose-dependent protection in oxidativeDNA damage Noroozi et al., 1998), it possesses preventive propertiesagainst damages of vascular and connective tissues (especially skin) andit is therapeutically useful in the treatment of dysfunctions of thedigestive tract (Seto et al. 1992).

Now we have found by experiments that the combination of vitamin C withthe most easily bioavailable bioflavonoid, isoquercetin, is mosteffective in prevention of and in defense against stress dysfunctions,especially against oxidative damages of living tissues including brain,vascular, connective tissues (especially skin).

It has been found that a composition containing ascorbic acid and one ormore derivates of quercetin elected from the groupquercetin-3-O-glucoside (isoquercetin), quercetin-4′-glucoside,quercetin-3′-glucoside and acid-quercetin-7-glucoside in a molar ratioof ascorbate to flavonoid in the range of 2:1 to 1:2, preferably in themolar ratio of 1:1, orally administered conveys in vivo higherprotection, longer maintenance of biological activity, higherconcentration in tissues and higher biological efficiency to vitamin Cin organs of human body. This adduct similarly also provides theproperties of higher protection, longer maintenance of biologicalactivity, higher concentration in tissues, and higher biologicalefficiency in organs of human body to isoquercetin and the otherglucosides of the above mentioned groups.

Useful compositions may contain in a daily dose 30–4000 mg of an activeamount of ascorbic acid or preferably of physiologically activeascorbate in form of its sodium salt, calcium, other mineral, or organiccation salts. Usually compositions contain 150–1000 mg, but for specialtreatments the amount is chosen higher between 1000 and 4000 mg,preferably between 1500 and 3000 mg. The compositions according to thepresent invention may be prepared in form of tablets, capsules orsyrups. These application forms may also contain further activeingredients in useful amounts like vitamins, suitable salts of Mg, Ca, Kor Fe and perhaps trace elements.

The compositions of the present invention preferably are useful as foodsupplements, but they may also be administered in a pharmaceuticaltreatment.

The present invention makes available

a) a method of maintaining long biological activity and highconcentration of ascorbate and isoquercetin in human organs (includingskin), tissues and cells,

b) a method of protection against oxidative damages of human organs,tissues, skin cells,

c) a method of prevention of arteriosclerosis, cardiovascular diseases,and other damages of vascular tissues, of allergic and inflammatorydisorders, of bacterial and viral infections, of metabolic dysfunctionsinvolving oxidative damages e.g., premature ageing,

d) a method of supporting pharmacological treatments of diseases anddysfunctions caused by oxidative damages,

by orally administration of a composition described above. Generallyspeaking, compositions that are applicable contain at least ascorbicacid or ascorbate or any other form of this vitamin that would in vivoyield ascorbate, or semidehydroascorbic acid, or dehydroascorbic acidand isoquercetin. The decision which further ingredients should becomponents of a composition useful in one of the above mentioned methodsdepends on the special indication. Usually, if the composition isadministered as a way of protection or prevention useful furtheringredients may be further vitamins, salts of Mg, Ca, K, Fe and traceelements in known amounts as used in food supplements. Compositionsuseful in method of supporting pharmacological treatments may differfrom them.

The superiority of isoquercetin and ascorbate used in combination forthe protection of human cells, tissues and organs from the oxidativestress is based on two properties of isoquercetin and of ascorbate.First, on the quick intestinal absorption of orally administeredisoquercetin and of ascorbate, and on the rapid and simple passage ofboth compounds through cytomembranes of human organs; secondly, on thespecificity of interaction of isoquercetin with ascorbate. Specifically,ascorbate maintains isoquercetin in its active oxidised state andisoquercetin maintains ascorbate in its enzymatically active reducedstate.

On the basis of our research on the bioavailability and on redoxproperties of isoquercetin and ascorbate it has been found that orallyadministered mixtures of isoquercetin and ascorbate are most effectivein protecting the organs (including skin), tissues, and cells from thechronic intracellular oxidative damages.

The uptake of isoquercetin into the human body is facilitated by thesodium-dependent glucose transport system. This type of transportoccurring in most animal species (Coady et al., 1990) is active duringthe uptake of pyranosides as for example described by Hediger for methylalpha-D-glucopyranoside (Hediger et al., 1987). The sodium-dependentglucose transport system in mammals was studied in many laboratories.Koepsell and Spangenberg (1994) characterised Na(+)-D-glucosecotransport in the intestine. It is a cotransporting system composed ofa set of two subunits: transport-mediating proteins transport-modulatingproteins. The first translocates the substrates and the secondaccelerates the V_(max) of the Transport. The susceptibility ofisoquercetin to be transported using the Na(+)-D-glucose cotransport issuggested to be determined by the manner in which a non-glucose moietyis linked to glucose. More information about this is given in a reviewof Olson and Pessin, 1996. Direct evidence that isoquercetine usessodium-dependent glucose transport pathway of the intestinalbrush-border membranes was obtained by Gee et al., 1998.

Also the uptake of ascorbate by human is caused by a sodium dependentglucose transport system. Interactions between glucose and ascorbatetransport activity have been demonstrated in many tissues and cells(Rumsey and Levine, 1998). Apparently ascorbate is absorbed in humanintestine by a sodium-dependent active transport system, although invitro about 10–20% of ascorbic acid moves into cells in the absence ofsodium (Kuo et al., 1997). The carrier proteins in the intestinal cellmembranes bind and transport the vitamin across the membrane to itsintracellular site of action. There are differences in transportkinetics, tissue specificity, Na⁺-dependence and energy dependence(Rumsey and Levine, 1.c.), but in most cases the transport of ascorbateis Na⁺-dependent and requires metabolic energy. Kinetic evidencesuggests strongly that ascorbate may be transported by the sametransporter as glucose and, therefore, by the same transporter asisoquercetin.

Pharmacokinetic studies with isoquercetin support the present inventionas they show excellent absorption rate and bioavailability ofisoquercetin. It is absorbed better than rutin and quercetin (Hollman,1997). Absorbed isoquercetin interacts with ascorbate protecting it and,at the same time, is being protected by ascorbate by being kept in thereduced state (Yamasaki et al., 1997). It has also been shown thatascorbate protects quercetin (aglycone of the isoquercetin) againstoxidative degradation and maintains quercetin's antiviral properties(Vrijsen et al., 1988). Effectiveness of isoquercetin in interactingwith ascorbate is strengthened by the fact that isoquercetin uses thepreferential intestinal Na(4+)-D-glucose cotransport discussed above.

Therefore, a most powerful dietary antioxidant composition is preparedusing among other ingredients ascorbic acid and isoquercetin. Theadvantageous properties of these compositions are induced by thesynergistic effect of isoquercetin protecting the activity of the orallyadministered ascorbic acid while maintaining its enzymatically activereduced form, and, on the other side, of ascorbate maintainingisoquercetin in its active oxidised state.

Surprisingly it was found that in contrast to other quercetinglucosides, isoquercetin shows far better absorption rates in humanintestinal tract than rutin or the quercetin aglycone and that it actsas a specific and most powerful dietary antioxidant at the same time.

This positive result was unexpected because mixtures of ascorbic acidand quercetin or quercetin glucosides other than isoquercetin wereconsiderably less effective.

Subject of this invention is that in humans the oral administration of amixture or combination of ascorbic acid and isoquercetin(quercetin-3-O-glucoside); or of any of mixtures of ascorbic acid andquercetin-4′-glucoside; of ascorbic acid and quercetin-3′-glucoside; ofascorbic acid and quercetin-7-glucoside, with a suitable molar ratio,preferably equimolar ratio, of ascorbate to flavonoid, conveys efficientprotection against oxidative damages, due to long maintenance ofbiological activity of each of the ingredients and due to maintenance ofhigh concentration of both ascorbate and isoquercetin in organs,tissues, and cells.

The invention of this application includes especially compositionscontaining the above mentioned ingredients useful for the prevention andtreatment of atherosclerosis and other cardiovascular disorders, certainforms of cancer, allergic and inflammatory disorders, bacterial andviral infections, a number of metabolic dysfunctions, e.g. prematureageing and other pathological conditions that involve oxidative damages.

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1. A method of extending the biological activity lifetime of ascorbicacid, ascorbate or a derivative thereof, comprising administeringascorbic acid, ascorbate or a derivative thereof in combination with oneor more of quercetin-3-O-glucoside (isoquercetin),quercetin-4′-glucoside, quercetin-3′-glucoside, orquercetin-7-glucoside, in a molar ratio of from about 2:1 to about 1:2.2. The method of claim 1, wherein said ascorbic acid or ascorbate ispresent in an amount ranging from 150 to 1000 mg in a daily dose.
 3. Amethod according to claim 1 wherein said isoquercetin is in combinationwith ascorbic acid or a physiologically active ascorbate in the form ofits sodium, calcium, or other mineral or organic salt.
 4. A methodaccording to claim 1 wherein said isoquercetin is in combination withascorbic acid or a mineral or organic salt thereof.
 5. A methodaccording to claim 1 wherein said combination further comprises avitamin.
 6. A method according to claim 1 wherein said combinationfurther comprises a Mg, Ca, K, or Fe salt.
 7. A method according toclaim 1 wherein said combination further comprises a trace element.
 8. Amethod according to claim 1 wherein said combination of ascorbic acid orascorbate and isoquercetin is in a molar ratio of about 1:1.
 9. A methodaccording to claim 1 wherein said ascorbic acid or ascorbate is presentin an amount ranging from 30 to 4000 mg in a daily dose.
 10. A methodaccording to claim 1 wherein said ascorbic acid or ascorbate is presentin an amount ranging from 1500 to 3000 mg in a daily dose.